GSM7658983: E10_3; Homo sapiens; RNA-Seq
11 days after transduction, cells were lysed in DNA lysis buffer (20mM Tris-HCl (pH 8.4), 50mM KCl, 0.45% NP40, 0.45% Tween-20, 10% Proteinase K) at 60°C for 1 hour. Samples lysed in DNA lysis buffer were denatured at 95º and amplified with a KAPA polymerase master mix (Roche, KK2602) PCR was performed in technical duplicates for each sample. Indexed primers containing Illumina flow-cell sequences were obtained from IDT (forward: 5'AATGATACGGCGACCACCGAGATCTACANNNNNNNNAAGGTGCTTCTCGATC TGCAT; reverse:5'CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCT where N represents the index nucleotides). Resulting products were confirmed to give single-band products of the expected size using gel electrophoresis and then pooled and purified for sequencing using the Zymo Select-a-Size DNA Clean & Concentrator kit (D4080). After pooling, the PCR product was quantified using the Qubit 3 Fluorometer. Samples were sequenced using Illumina HiSeq 2500 Rapid Run technology. Briefly, samples were loaded onto the flow cell at a final concentration of 10pM with a 20% PhiX spike-in due to low sequence diversity. Sequencing was run for 50 cycles with single-read setting. Raw sequencing reads in fastQ format were first processed to generate a table of cell-line barcode counts for each well, and barcode counts were then filtered for in-set cell lines.